Despite advances in the therapy of opportunistic infections complicating AIDS, they remain a leading cause of morbidity and mortality. New agents are needed to combat infections by Pneumocystis carinii, Toxoplasma gondii and Mycobacterium tuberculosis and avium. We are in the process of cloning, expressing and characterizing three enzymes from these organisms to provide in vitro targets for the development of new drugs; these enzymes are dihydrofolate reductase (DHFR), thymidylate synthase (TS), and dihydropteroate synthase (DHPS). We will compare the interactions of inhibitors with P. carinii and human DHFR using binding studies and site directed mutagenesis guided by molecular modeling. The objective is to identify the molecular interactions which lead to species discrimination of inhibition. We will also prepare 15N-13C labeled Pneumocystis DHFR for NMR structural studies in collaboration with a collaborator. Toxoplasma TS-DHFR will be expressed, purified and characterized, and the RS and DHFR domains will be separated by manipulation of the gene to facilitate structural characterization. Mycobacterial TS and DHFR clones will be sequenced and the enzymes will be expressed in E. coli. Clones encoding DHPS from Toxoplasma and Mycobacterium will be isolated from cDNA and genomic DNA libraries. These and the Pneumocystis DHPS will be expressed, purified and characterized. Expression of enzymes will be achieved by subcloning coding sequences into E. coli or yeast expression systems. The enzymes will be purified using specific affinity systems when available or by combinations of conventional chromatography. DHFRs will be screened with existing inhibitors. We have established collaborations for x-ray crystallographic structural determinations. The availability of in vitro enzyme systems is providing a valuable resource for the identification and design of new inhibitors of de novo thymidylate synthesis and folate metabolism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032784-07
Application #
2429405
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1991-09-01
Project End
1999-05-31
Budget Start
1997-06-01
Budget End
1998-05-31
Support Year
7
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Trujillo, M; Duncan, R; Santi, D V (1997) Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme. Protein Eng 10:567-73
Trujillo, M; Donald, R G; Roos, D S et al. (1996) Heterologous expression and characterization of the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of Toxoplasma gondii. Biochemistry 35:6366-74
Livi, L L; Edman, U; Schneider, G P et al. (1994) Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans. Gene 150:221-6
Margosiak, S A; Appleman, J R; Santi, D V et al. (1993) Dihydrofolate reductase from the pathogenic fungus Pneumocystis carinii: catalytic properties and interaction with antifolates. Arch Biochem Biophys 305:499-508
Santi, D V; Edman, U; Minkin, S et al. (1991) Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase. Protein Expr Purif 2:350-4